miR-17-92 cluster-BTG2 axis regulates B-cell receptor signaling in mantle cell lymphoma.

B-cell receptor (BCR) signaling is critically activated and stable for mantle cell lymphoma (MCL), but the underlying mechanism of the activated BCR signaling pathway is not clear. The pathogenic basis of miR-17-92 cluster remains unclear although the oncogenic microRNA (miRNA) miR-17-92 cluster is highly expressed in patients with MCL. We revealed that miR-17-92 cluster overexpression is partly dependent on SOX11 expression and chromatin acetylation of MIR17HG enhancer regions. Moreover, miR-17-92 cluster regulates not only cell proliferation but BCR signaling activation in MCL cell lines. To comprehensively identify miR-17-92 cluster target genes, we performed pulldown-seq, where target RNA of miRNA was captured using the biotinylated miRNA mimics and magnetic bead-coated streptavidin, and quantified using next-generation sequencing. The pulldown-seq identified novel miRNA target genes, including tumor suppressors such as BTG2 (miR-19b), CDKN2A (miR-17), SYNE1 (miR-20a), TET2 (miR-18, miR-19b, and miR-92a), TNFRSF10A (miR-92a), and TRAF3 (miR-17). Notably, the gene expression profile data of patients with MCL revealed that BTG2 expression was negatively associated with that of BCR signature genes, and low BTG2 expression was associated with poor overall survival. Moreover, BTG2 silencing in MCL cell lines significantly induced BCR signaling overactivation and cell proliferation. Our results suggest an oncogenic role of miR-17-92 cluster-activating BCR signaling throughout BTG2 deregulation in MCL. Furthermore, this may contribute to the prediction of the therapeutic efficacy and improved outcomes of MCL.

A Novel CD135+ Subset of Mouse Monocytes with a Distinct Differentiation Pathway and Antigen-Presenting Properties.

The mononuclear phagocyte system (MPS), composed of monocytes/macrophages and dendritic cells (DCs), plays a critical role at the interface of the innate and adaptive immune systems. However, the simplicity of MPS has been challenged recently by discoveries of novel cellular components. In the current study, we identified the CD135+ subset of monocytes as a novel class of APCs in mice. CD135+ monocytes were readily found in the bone marrow, spleen, and peripheral blood at steady state, and they expressed markers specific to DCs, including MHC class II and CD209a, along with markers for monocytes/macrophages. In addition, this subset phagocytosed bacteria and activated naive T lymphocytes, fulfilling the criteria for APCs. CD135+ monocytes were derived directly from macrophage DC progenitors, not from common monocyte progenitors or other monocytes, suggesting that these are distinct from conventional monocytes. These findings facilitate our understanding of the MPS network that regulates immune responses for host defense.

Case study observational research: inflammatory cytokines in the bronchial epithelial lining fluid of COVID-19 patients with acute hypoxemic respiratory failure.

BACKGROUND: In this study, the concentrations of inflammatory cytokines were measured in the bronchial epithelial lining fluid (ELF) and plasma in patients with acute hypoxemic respiratory failure (AHRF) secondary to severe coronavirus disease 2019 (COVID-19). METHODS: We comprehensively analyzed the concentrations of 25 cytokines in the ELF and plasma of 27 COVID-19 AHRF patients. ELF was collected using the bronchial microsampling method through an endotracheal tube just after patients were intubated for mechanical ventilation. RESULTS: Compared with those in healthy volunteers, the concentrations of interleukin (IL)-6 (median 27.6 pmol/L), IL-8 (1045.1 pmol/L), IL-17A (0.8 pmol/L), IL-25 (1.5 pmol/L), and IL-31 (42.3 pmol/L) were significantly greater in the ELF of COVID-19 patients than in that of volunteers. The concentrations of MCP-1 and MIP-1ß were significantly greater in the plasma of COVID-19 patients than in that of volunteers. The ELF/plasma ratio of IL-8 was the highest among the 25 cytokines, with a median of 737, and the ELF/plasma ratio of IL-6 (median: 218), IL-1ß (202), IL-31 (169), MCP-1 (81), MIP-1ß (55), and TNF-α (47) were lower. CONCLUSIONS: The ELF concentrations of IL-6, IL-8, IL-17A, IL-25, and IL-31 were significantly increased in COVID-19 patients. Although high levels of MIP-1 and MIP-1ß were also detected in the blood samples collected simultaneously with the ELF samples, the results indicated that lung inflammation was highly compartmentalized. Our study demonstrated that a comprehensive analysis of cytokines in the ELF is a feasible approach for understanding lung inflammation and systemic interactions in patients with severe pneumonia.

7,8-Dihydroxy-3-(4'-hydroxyphenyl)coumarin inhibits invasion and migration of osteosarcoma cells.

Advances in pharmacy and medicine have led to the development of many anti-cancer and molecular targeted agents; however, there are few agents capable of suppressing metastasis. To prevent cancer recurrence, it is essential to develop novel agents for inhibiting metastasis. Coumarin-based compounds have multiple pharmacological activities including anti-cancer effects. We screened a compound library constructed at Kyoto Pharmaceutical University and showed that 7,8-dihydroxy-3-(4'-hydroxyphenyl)coumarin (DHC) inhibited invasion and migration of LM8 mouse osteosarcoma cells and 143B human osteosarcoma cells in a concentration-dependent manner. DHC decreased intracellular actin filament formation by downregulating Rho small GTP-binding proteins such as RHOA, RAC1, and CDC42, which regulate actin reorganization. However, DHC did not downregulate the corresponding mRNA transcripts, whereas it downregulated Rho small GTP-binding proteins in the presence of cycloheximide, suggesting that DHC enhances the degradation of these proteins. DHC treatment inhibited metastasis and prolonged overall survival in a spontaneous metastasis mouse model. These results indicate that DHC has the potential to suppress metastasis of osteosarcoma cells by downregulating Rho small GTP-binding proteins.

Association of the CYP39A1 G204E Genetic Variant with Increased Risk of Glaucoma and Blindness in Patients with Exfoliation Syndrome.

PURPOSE: Carriers of functionally deficient mutations in the CYP39A1 gene have been recently reported to have a 2-fold increased risk of exfoliation syndrome (XFS). The aim of this study was to evaluate the risk of blindness and related clinical phenotypes of XFS patients carrying the loss-of-function CYP39A1 G204E mutation in comparison with XFS patients without any CYP39A1 mutation. DESIGN: Retrospective case study. PARTICIPANTS: A total of 35 patients diagnosed with XFS carrying the CYP39A1 G204E mutation and 150 XFS patients without any CYP39A1 mutation who were randomly selected from the Japanese XFS cohort. METHODS: Two-sided Fisher exact test with an alpha level < 0.05 was used to estimate the significance of the calculated odds ratio (OR) for all categorical measures. Comparisons between groups of subjects were performed using linear mixed effect models with group as random effect and taking possible dependence between eyes within a subject into account. MAIN OUTCOME MEASURES: Primary analysis compared the incidence of blindness (defined as visual acuity [VA] < 0.05 decimal), prevalence of exfoliation glaucoma (XFG), history of glaucoma surgery, and indices of glaucoma severity such as visual field (VF) mean deviation (MD), intraocular pressure (IOP), and vertical cup-disc ratio (CDR) between CYP39A1 G204E carriers and those without any CYP39A1 mutation. RESULTS: The overall risk for blindness was significantly higher in XFS patients carrying the CYP39A1 G204E variant (10/35 [28.6%]) compared with XFS patients without any CYP39A1 mutations (8/150 [5.4%]; odds ratio [OR], 7.1; 95% confidence interval [CI], 2.7-20.2]; P < 0.001). A higher proportion of XFS patients with the CYP39A1 G204E mutation (23/35 [65.7%]) had evidence of XFG in at least 1 eye compared with the comparison group (41/150 [27.3%]; OR, 5.1; 95% CI, 2.4-11.4]; P < 0.0001). Significantly higher peak IOP, larger vertical CDR, and worse VF MD were also found in CYP39A1 G204E variant carriers (P < 0.001). Additionally, patients with the CYP39A1 G204E mutation (18/35 [51.4%]) required more laser or glaucoma surgical interventions compared with those without any CYP39A1 mutation (32/150 [21.3%], P < 0.001). CONCLUSIONS: Patients with XFS carrying the CYP39A1 G204E mutation had significantly increased risk of blindness, higher occurrence of XFG, and more severe glaucoma compared with patients with XFS without any CYP39A1 mutation.

Aberrant astrocyte Ca2+ signals "AxCa signals" exacerbate pathological alterations in an Alexander disease model.

Alexander disease (AxD) is a rare neurodegenerative disorder caused by gain of function mutations in the glial fibrillary acidic protein (GFAP) gene. Accumulation of GFAP proteins and formation of Rosenthal fibers (RFs) in astrocytes are hallmarks of AxD. However, malfunction of astrocytes in the AxD brain is poorly understood. Here, we show aberrant Ca2+ responses in astrocytes as playing a causative role in AxD. Transcriptome analysis of astrocytes from a model of AxD showed age-dependent upregulation of GFAP, several markers for neurotoxic reactive astrocytes, and downregulation of Ca2+ homeostasis molecules. In situ AxD model astrocytes produced aberrant extra-large Ca2+ signals "AxCa signals", which increased with age, correlated with GFAP upregulation, and were dependent on stored Ca2+ . Inhibition of AxCa signals by deletion of inositol 1,4,5-trisphosphate type 2 receptors (IP3R2) ameliorated AxD pathogenesis. Taken together, AxCa signals in the model astrocytes would contribute to AxD pathogenesis.

Stage-specific reference genes significant for quantitative PCR during mouse retinal development.

Developing mouse retina has been serving as an ideal model for investigating the molecular mechanism of neural development and angiogenesis, because several significant events associated with these physiological phenomena are drastically occurring in conjunction with retinal development. However, as many genes are influencing on each other to establish mature retina within 21 days from E10 to P12, we must carefully design the experiments, such as in the case of quantitating the amount of altered gene expression toward the establishment of retina by quantitative PCR. As we have seen considerable variations of quantitative results in different developmental stages of retina depending on the reference genes used for compensation, we here attempted to determine a reliable reference gene to accurately quantitate the target genes in each stage. According to the results of in silico prediction and comparison with a database of SAGE, we found that the most stable gene from early to late stages was Sdha, whereas one of the most popular housekeeping genes, Actb, was the one that could mislead the quantitative results even in the adult stage. Consequently, we pointed out the importance of selecting an appropriate reference gene, especially to quantitate the amount of gene expression in the developmental stages of a certain tissue.

Dysregulation of the TCF4 Isoform in Corneal Endothelial Cells of Patients With Fuchs Endothelial Corneal Dystrophy.

Purpose: This study evaluated the dysregulation of TCF4 isoforms and differential exon usage (DEU) in corneal endothelial cells (CECs) of Fuchs endothelial corneal dystrophy (FECD) with or without trinucleotide repeat (TNR) expansion in the intron region of the TCF4 gene. Methods: Three RNA-Seq datasets of CECs (our own and two other previously published datasets) derived from non-FECD control and FECD subjects were analyzed to identify TCF4 isoforms and DEU events dysregulated in FECD by comparing control subjects to those with FECD with TNR expansion and FECD without TNR expansion. Results: Our RNA-Seq data demonstrated upregulation of three TCF4 isoforms and downregulation of two isoforms in FECD without TNR expansion compared to the controls. In FECD with TNR expansion, one isoform was upregulated and one isoform was downregulated compared to the control. Additional analysis using two other datasets identified that the TCF4-277 isoform was upregulated in common in all three datasets in FECD with TNR expansion, whereas no isoform was dysregulated in FECD without TNR expansion. DEU analysis showed that one exon (E174) upstream of the TNR, which only encompassed TCF4-277, was upregulated in common in all three datasets, whereas eight exons downstream of the TNR were downregulated in common in all three datasets in FECD with TNR expansion. Conclusions: This study identified TCF4-277 as a dysregulated isoform in FECD with TNR expansion, suggesting a potential contribution of TCF4-277 to FECD pathophysiology.

Potential marker subset of blood-circulating cytokines on hematopoietic progenitor-to-Th1 pathway in COVID-19.

In this study, we analyzed a relatively large subset of proteins, including 109 kinds of blood-circulating cytokines, and precisely described a cytokine storm in the expression level and the range of fluctuations during hospitalization for COVID-19. Of the proteins analyzed in COVID-19, approximately 70% were detected with Bonferroni-corrected significant differences in comparison with disease severity, clinical outcome, long-term hospitalization, and disease progression and recovery. Specifically, IP-10, sTNF-R1, sTNF-R2, sCD30, sCD163, HGF, SCYB16, IL-16, MIG, SDF-1, and fractalkine were found to be major components of the COVID-19 cytokine storm. Moreover, the 11 cytokines (i.e., SDF-1, SCYB16, sCD30, IL-11, IL-18, IL-8, IFN-γ, TNF-α, sTNF-R2, M-CSF, and I-309) were associated with the infection, mortality, disease progression and recovery, and long-term hospitalization. Increased expression of these cytokines could be explained in sequential pathways from hematopoietic progenitor cell differentiation to Th1-derived hyperinflammation in COVID-19, which might also develop a novel strategy for COVID-19 therapy with recombinant interleukins and anti-chemokine drugs.

Proteomic pattern analysis discriminates among multiple sclerosis-related disorders.

OBJECTIVE: To use a new, unbiased biomarker discovery strategy to obtain and assess proteomic data from cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS)-related disorders. METHODS: CSF protein profiles were analyzed from 107 patients with either MS-related disorders (including relapsing remitting MS [RRMS], primary progressive MS [PPMS], anti-aquaporin4 antibody seropositive-neuromyelitis optica spectrum disorder [SP-NMOSD], and seronegative-NMOSD with long cord lesions on spinal magnetic resonance imaging [SN-NMOSD]), amyotrophic lateral sclerosis (ALS), or other inflammatory neurological diseases (used as controls). CSF peptides/proteins were purified with magnetic beads, and directly measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The obtained spectra were analyzed with multivariate statistics and pattern matching algorithms. These analyses were replicated in an independent sample set of 84 patients composed of those with MS-related disorders or with other neurological diseases (the second cohort). RESULTS: MS-related disorders differed considerably in terms of CSF protein profiles. SP-NMOSD and SN-NMOSD, both of which fit within the NMO spectrum, were distinguishable from RRMS with high cross-validation accuracy on a support vector machine classifier, especially in relapse phases. Some peaks derived from samples of relapsed SP-NMOSD can discriminate RRMS with high area under curve scores (>0.95) and this was reproduced on the second cohort. The similarity of proteomic patterns between selected neurological diseases were demonstrated by pattern matching analysis. To our surprise, the spectral differences between RRMS and PPMS were much larger than those of PPMS and ALS. INTERPRETATION: Our findings suggest that CSF proteomic pattern analysis can increase the accuracy of disease diagnosis of MS-related disorders and will aid physicians in appropriate therapeutic decision-making.

RNA-Seq-based transcriptome analysis of corneal endothelial cells derived from patients with Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is the most common inherited corneal disease. Fibrillar focal excrescences called guttae and corneal edema due to corneal endothelial cell death result in progressive vision loss. Multiple genetic variants have been reported, but the pathogenesis of FECD is not fully understood. In this study, we used RNA-Seq to analyze differential gene expression in the corneal endothelium obtained from patients with FECD. Differential expression analysis of transcriptomic profiles revealed that expression of 2366 genes (1092 upregulated and 1274 downregulated genes) was significantly altered in the corneal endothelium of patients with FECD compared to healthy subjects. Gene ontology analysis demonstrated an enrichment of genes involved in extracellular matrix (ECM) organization, response to oxidative stress, and apoptotic signaling. Several pathway analyses consistently indicated the dysregulation of ECM-associated pathways. Our differential gene expression findings support the previously proposed underlying mechanisms, including oxidative stress and apoptosis of endothelial cells, as well as the phenotypic clinical FECD hallmark of ECM deposits. Further investigation focusing on differentially expressed genes related to these pathways might be beneficial for elucidating mechanisms and developing novel therapies.

Novel Vertical Cup-to-Disc Classification to Identify Normal Eyes That Maintain Non-Glaucoma Status: A 10-Year Longitudinal Study.

PRCIS: We propose a new classification model to serve as a control for future genomic studies of glaucoma by distinguishing normal subjects maintaining non-glaucoma status for 10 years using the vertical cup-to-disc ratio (VCDR). PURPOSE: This study aimed to develop a classification for distinguishing subjects maintaining non-glaucoma status for 10 years using the VCDR. PARTICIPANTS AND METHODS: Among 842 volunteers 40 years and older, 421 volunteers participated in the second ophthalmic examination 10 years after their first examination. Each volunteer was diagnosed either as healthy normal or glaucoma suspect (GS) in the first glaucoma screening examinations. The former was further classified into the 3 grades of N1, N2, and N3. Specifically, N1 represented (1) VCDR <0.3; (2) no notching or nerve fiber layer defect; and (3) no undermining, N2 indicated 0.3≤VCDR<0.6 and conditions (2) and (3) of N1; and N3 represented 0.3≤VCDR<0.6 with undermining and condition (2), or 0.6≤VCDR<0.7 and condition (2) of N1. Glaucoma transition rates (GTRs) were evaluated in 421 volunteers who returned to participate after a 10-year period. RESULTS: GTRs were calculated as 1.3% in both N1 and N2, 3.9% in N3, and 18.2% in GS. The ratio of volunteers in the same category maintenance rate increased from N1 to N3. CONCLUSION: GTRs were lower in N1 and N2 than in N3 or GS during the 10-year study period. This novel classification of healthy non-glaucoma subjects may help identify those, especially Japanese males, who maintain a non-glaucoma status for an extended period of 10 years.

Cerebrospinal fluid proteomic patterns discriminate Parkinson's disease and multiple system atrophy.

The differential diagnosis of Parkinson's disease and multiple system atrophy can be challenging, especially in the early stages of the diseases. We developed a proteomic profiling strategy for parkinsonian diseases using mass spectrometry analysis for magnetic-bead-based enrichment of cerebrospinal fluid peptides/proteins and subsequent multivariate statistical analysis. Cerebrospinal fluid was obtained from 37 patients diagnosed with Parkinson's disease, 32 patients diagnosed with multiple system atrophy, and 26 patients diagnosed with other neurological diseases as controls. The samples were from the first cohort and the second cohort. Cerebrospinal fluid peptides/proteins were purified with C8 magnetic beads, and spectra were obtained by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Principal component analysis and support vector machine methods are used to reduce dimension of the data and select features to classify diseases. Cerebrospinal fluid proteomic profiles of Parkinson's disease, multiple system atrophy, and control were differentiated from each other by principal component analysis. By building a support vector machine classifier, 3 groups were classified effectively with good cross-validation accuracy. The model accuracy was well preserved for both cases, training by the first cohort and validated by the second cohort and vice versa. Receiver operating characteristics proved that the peak of m/z 6250 was the most important to differentiate multiple system atrophy from Parkinson's disease, especially in the early stages of the disease. A proteomic pattern classification method can increase the accuracy of clinical diagnosis of Parkinson's disease and multiple system atrophy, especially in the early stages.

Three susceptible loci associated with primary open-angle glaucoma identified by genome-wide association study in a Japanese population.

Primary open-angle glaucoma (POAG) is the major type of glaucoma. To discover genetic markers associated with POAG, we examined a total of 1,575 Japanese subjects in a genome-wide association study (stage 1) and a subsequent study (stage 2). Both studies were carried out at a single institution. In the stage 1 association study, we compared SNPs between 418 POAG patients and 300 control subjects. First, low-quality data were eliminated by a stringent filter, and 331,838 autosomal SNPs were selected for analysis. Poorly clustered SNPs were eliminated by a visual assessment, leaving 255 that showed a significant deviation (P < 0.001) in the allele frequency comparison. In the stage 2 analysis, we tested these 255 SNPs for association in DNA samples from a separate group of 409 POAG and 448 control subjects. High-quality genotype data were selected and used to calculate the combined P values of stages 1 and 2 by the Mantel-Haenszel test. These analyses yielded 6 SNPs with P < 0.0001. All 6 SNPs showed a significant association (P < 0.05) in stage 2, demonstrating a confirmed association with POAG. Although we could not link the SNPs to the annotated gene(s), it turned out that we have identified 3 genetic loci probably associated with POAG. These findings would provide the foundation for future studies to build on, such as for the metaanalysis, to reveal the molecular mechanism of the POAG pathogenesis.

Efficient DNA/RNA extraction from tarsal plates by SK mill, a freeze-crush apparatus.

The tarsal plate is an eyelid tissue that maintains lid structure from inside the upper/lower eyelids, and it surrounds the meibomian glands and supports their unique secretion mechanism. Sebaceous carcinoma, a malignant eyelid tumour, can sometimes develop from the meibomian glands and is usually excised together with the tarsal plate during surgery, so the tarsal plate serves as a control research tissue. However, since the plate is thick, hard and heterogeneous with few cells, obtaining enough genomic DNA and/or total RNA is often difficult. Therefore, we attempted to establish an efficient protocol to obtain DNA and RNA simultaneously by comparing the combinations of homogenization (mortar/pestle, pellet pestle or SK mill) and purification (organic solvent or spin column) methods using rabbit tarsal plates. Based on the yield, quality and hands-on time, the SK mill and spin column was found to be the most efficient combination. We then applied the established protocol to extract DNA/RNA from six human tarsal-plate samples and succeeded in generating high-quality exome and transcriptome datasets via a next-generation sequencer with sufficient coverage and meibomian gland-specific expression of representative genes, respectively. Our new findings will provide ideal reference data for future genetic and gene-expression studies of sebaceous carcinoma.

Seasonal Variation and Trend of Intraocular Pressure Decrease Over a 20-Year Period in Normal-Tension Glaucoma Patients.

PURPOSE: To investigate the trend of seasonal variation of intraocular pressure (IOP) in patients with normal-tension glaucoma over a 20-year period by retrospectively analyzing the Kyoto Prefectural University of Medicine Glaucoma Registry database as real-world data. DESIGN: Retrospective cohort study. METHODS: Data points (n = 49,007) were extracted retrospectively from the medical records of 1774 patients with normal-tension glaucoma (665 male patients and 1109 female patients; mean ± SD age was 59.8 ± 14.4 years; and mean ± SD observation period was 5.6 ± 4.4 years) seen over the 20-year period. We first calculated the mean IOP from all available data of each month from January 1997 through December 2016. The data were then categorized into 5 groups of 4 consecutive years each (1997-2000, 2001-2004, 2005-2008, 2009-2012, and 2013-2016) and the mean IOP of each month within the group was calculated. Seasonal variations of IOP over the 20-year study period and in the 5 consecutive groups were then investigated via nonlinear multiple regression analysis. RESULTS: A continuous decrease of IOP was detected throughout the 20-year period (P < .001), with distinct seasonal variation. The annual mean ± SD IOP was highest (13.9 ± 2.7 mm Hg) in the oldest group (1997-2000), with a gradual decrease in each subsequent group, finally becoming lowest (12.3 ± 2.7 mm Hg) in the most recent group (2013-2016) (P < .001), and all of them were accompanied by distinct seasonal variation (P < .001). CONCLUSIONS: Based on the Kyoto Prefectural University of Medicine Glaucoma Registry real-world longitudinal data, our findings revealed a continuous decrease and distinct seasonal variation of IOP in patients with normal-tension glaucoma throughout the 20-year study period.

Association between prostaglandin E receptor 3 polymorphisms and Stevens-Johnson syndrome identified by means of a genome-wide association study.

BACKGROUND: Stevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), are acute inflammatory vesiculobullous reactions of the skin and mucosa. They often affect the ocular surface and can result in permanent visual dysfunction. OBJECTIVES: We sought to discover genetic markers for SJS/TEN susceptibility. METHODS: We performed a genome-wide association study with 60 patients and 300 control subjects. We applied stringent filter and visual assessments for selecting single nucleotide polymorphisms (SNPs) and a high false discovery rate threshold. We fine-mapped the region where a candidate SNP was found and confirmed the results by means of sequencing. We evaluated the function of agonist-activated prostaglandin E receptor 3 (EP3), the gene for which contained several SNPs, in regulating cytokine production in human conjunctival epithelial (CE) cells. The expression levels of EP3 in the CE cells from patients and control subjects were also compared. RESULTS: We identified 3 SNPs that passed the false discovery rate threshold. One (rs17131450) was close to the EP3 gene. Therefore we analyzed the EP3 region in detail and identified 5 other SNPs. We confirmed the association between SJS/TEN and all 6 SNPs. Activated EP3 was expressed in control CE cells, and it suppressed polyI:C-stimulated cytokine production, suggesting that EP3 might help prevent ocular surface inflammation. Concordantly, the EP3 levels were much lower in the CE cells of the patients than in those of the control subjects. CONCLUSION: We demonstrated, using both genetic and functional analyses, that EP3 could be a key player in the pathogenesis of SJS/TEN accompanied by ocular complications.

Detection of novel and recurrent conjoined genes in non-Hodgkin B-cell lymphoma.

For this study, we investigated comprehensive expression of conjoined genes (CGs) in non-Hodgkin B-cell lymphoma (B-NHL) cell line KPUM-UH1 by using paired-end RNA sequencing. Furthermore, we analyzed the expression of these transcripts in an additional 21 cell lines, 37 primary samples of various malignancies and peripheral blood mononuclear cells of four normal individuals. Seventeen CGs were detected in KPUM-UH1: CTBS-GNG5, SRP9-EPHX1, RMND5A-ANAPC, OTX1-EHBP1, ATF2-CHN1, PRKAA1-TTC33, LARP1-MRPL22, LOC105379697-BAK1, TIAM2-SCAF8, SPAG1-VPS13B, WBP1L-CNNM2, NARS2-GAB2, CTSC-RAB38, VAMP1-CD27-AS1, LRRC37A2-NSF, UBA2-WTIP and ZNF600-ZNF611. To our knowledge, 10 of these genes have not been previously reported. The various characteristics of the CGs included in- and out-of-frame fusions, chimeras involving non-coding RNA and transcript variants. A finding of note was that LARP1-MRPL2 was characterized as in-frame fusion and was recurrently expressed in B-NHL samples. In this study, variety of CGs was expressed both in malignant and normal cells, some of which might be specific to lymphoma.

Transcriptome dataset of human corneal endothelium based on ribosomal RNA-depleted RNA-Seq data.

The corneal endothelium maintains corneal transparency; consequently, damage to this endothelium by a number of pathological conditions results in severe vision loss. Publicly available expression databases of human tissues are useful for investigating the pathogenesis of diseases and for developing new therapeutic modalities; however, databases for ocular tissues, and especially the corneal endothelium, are poor. Here, we have generated a transcriptome dataset from the ribosomal RNA-depleted total RNA from the corneal endothelium of eyes from seven Caucasians without ocular diseases. The results of principal component analysis and correlation coefficients (ranged from 0.87 to 0.96) suggested high homogeneity of our RNA-Seq dataset among the samples, as well as sufficient amount and quality. The expression profile of tissue-specific marker genes indicated only limited, if any, contamination by other layers of the cornea, while the Smirnov-Grubbs test confirmed the absence of outlier samples. The dataset presented here should be useful for investigating the function/dysfunction of the cornea, as well as for extended transcriptome analyses integrated with expression data for non-coding RNAs.

Novel use of a chemically modified siRNA for robust and sustainable in vivo gene silencing in the retina.

Despite efficient and specific in vitro knockdown, more reliable and convenient methods for in vivo knockdown of target genes remain to be developed particularly for retinal research. Using commercially available and chemically modified siRNA so-called Accell siRNA, we established a novel in vivo gene silencing approach in the rat retina. siRNA designed for knockdown of the house keeping gene Gapdh or four retinal cell type-specific genes (Nefl, Pvalb, Rho and Opn1sw) was injected into the vitreous body, and their retinal mRNA levels were quantified using real-time PCR. Intravitreal injection of siRNA for Gapdh resulted in approximately 40-70% reduction in its retinal mRNA levels, which lasted throughout a 9-day study period. Furthermore, all the selected retinal specific genes were efficiently down-regulated by 60-90% following intravitreal injection, suggesting injected siRNA penetrated into major retinal cell types. These findings were consistent with uniform distribution of a fluorescence-labeled siRNA injected into the vitreous body. Interestingly, gene silencing of Grin1, a core subunit of NMDA receptor, was accompanied by significant prevention from NMDA-induced retinal ganglion cell death. Thus, we provide single intravitreal injection of Accell siRNA as a versatile technique for robust and sustainable in vivo retinal gene silencing to characterize their biological functions under physiological and pathophysiological conditions.